【作者】 张连茹；

【导师】 邹国林；

【作者基本信息】 武汉大学 ， 生化及分子生物学， 2004， 博士

【摘要】 二色补血草Limonium bicolor Kunze(Bge.)是白花丹科(Plumbaginaceae)，补血草属(Limoniums)植物中的一种。又名燎眉蒿、扫帚草、匙叶草、血见愁、秃子花、苍蝇花、白花菜棵。属于多年生草本，稀一年生。散生于草原、草甸草原及山地，能适应沙质土及轻盐，归于草原旱生杂草类草。主要分布于东北、黄河流域诸省区及江苏北部，蒙古和西伯利亚也有。中药以其带根全草入药(亦入蒙药)，能补血、止血、温中健脾、滋补强壮，主治月经不调、功能性子宫出血、宫颈癌、肾盂肾炎、尿血、诸症体弱等。药理实验表明该属植物具有止血、抗病毒、抑菌、抗氧化等活性作用。 国外对该属植物的研究始于50年代，其化学成分以黄酮为主，根中多富含鞣质，常作为强壮剂和收敛剂，许多种是闻名的鞣料植物和染料植物。60年代末期曾以黄酮类化合物作为补血草属植物分类的依据。目前仍有关于从该属植物中发现新的化学成分以及相关生物活性的研究报道。但关于二色补血草的研究，尚未见报道。 国内对该属植物化学成分及药效的研究集中于80年代末90年代初，研究的重点是黄酮等次生代谢产物的分离提取。关于二色补血草仅有的研究包括王秉文等人(1988，1994)对其水提物及醇提物的止血作用和止血机理进行了研究；朱蓉等人(1991)对其化学成分进行了初步的研究，报道显示从二色补血草的地上部位的乙醇提取物中，共分离到5种已知的化合物(没食子酸、北美圣草素、木犀草素、槲皮素及杨梅素-没食子酰基-半乳糖苷)。 目前为止尚无对二色补血草水溶性成分如多糖的研究;亦没有关于二色补血草中特有的化合物的研究报道:在生物学活性方面缺乏对抗癌、抗氧化等方面的研究;缺乏相关的构效关系的研究;在研究开发方面:目前尚没有分离到一个可供开发的药效成分。 中药二色补血草在煎煮入药时，许多水溶性的有效成分进入水相，煎煮过程中也会产生新成分，比如配位络合物、分子络合物就是其中的主要成员。因而分离提取水相中的有效成分，研究其与金属离子等的络合作用，对确立中药二色补血草的药效成分，了解药用机理具有重要的意义。同时分离提取有机相中的化学成分，并进行结构解析，以期获得新的有效成分或做为先导化合物，为新药的研究开发提供理论基础。 本文在前人工作的基础上，分别对二色补血草的水相和有机相中的几个有效成分(水溶性多糖、多酚类化合物、挥发性成分)进行分离提取、结构表征，并进行了相关的生物学活性(抗肿瘤、抗菌、抗氧化、络合作用等)研究，同时对分离、纯化及分析方法进行了优化。希望为确定二色补血草的药效成分，开发中草药资源提供研究基础。主要内容包括:1总结了近年来关于中草药多糖、多酚类化合物(黄酮类)以及挥发油的分离纯化、结构分析、生物活性及构效关系等方面的研究进展。2从二色补血草中分离提取到具有体外抗肿瘤作用和抗氧化作用的二色补血草多糖LP，并通过波谱、元素分析等方法确定了其化学结构。本实验结果提示某些a一构型的葡聚糖也具有抗肿瘤活性。有关多糖的结构确定及抗肿瘤、抗氧化等生物效应日益受到人们的关注。而二色补血草多糖的研究尚属于空白。用乙醇脱脂，热水浸煮等方法对二色补血草的水溶性成分进行分离提取，经凝胶柱层析等方法纯化，得到了二色补血草水溶性多糖LP。红外光谱、凝胶渗透色谱(GPC)及元素分析等结果证明LP是不含N元素的均一多糖。气相色谱一质谱、纸层析、红外分析等方法确定其单糖基组成为葡萄糖和葡萄糖醛酸。GPC结果表明廿的分子量为4.63x103。综合’3C NMR、‘H NMR等分析结果确定LP的结构为:以a一D一【GlcA(1*6)Glc]二糖为结构单位，每个重复的二糖单位彼此以a一(1*4)糖普键连接成的直链多糖。其部分结构式如下:”4)一a一D一GlcA一(1叶6)一a一D一Gle一(l神。 用邻苯三酚自氧化法评价了LP的体外抗氧化作用，结果表明LP对邻苯三酚自氧化过程中产生的氧自由基有清除作用;用荧光光谱法考察了LP对金属锌离子的络合作用，LP的荧光峰出现在460nm附近，zn2+、ca2+通过与LP络合，使其荧光强度增加，荧光光谱的变化的结果表明LP对锌等金属离子具有络合作用;用MTr法评价了LP对HeLa细胞的细胞毒作用，其IC50为62.如岁mL，表明LP在体外对Heu细胞具有较强的抑制作用。一般认为大多数抗肿瘤活性的多糖具有1*3一B一D一葡聚糖结构，且往往带有0一6位的分枝，如香菇多糖等。本实验结果提示某些a一构型的葡聚糖也具有抗肿瘤活性。水溶性的二色补血草多糖很可能是二色补血草治疗宫颈癌的疗效成分。此外也考察了LP与BSA的相互作用及其对细胞溶血性的影响。3从二色补血草中分离提取到二种新的具有荧光特性的黄酮类化合物。二色补血草分别用正己烷、氯仿、氯仿:甲醇、甲醇提取，从其甲醇提取物中分离到没食子酸甲酷、芹菜素和二种新的黄酮类化合物，经NMR、IR、EI一MS等波谱学鉴定这两种新化合物分别为黄烯(5，7，4‘一trihydroxy一4一methal一flavene)和黄烷醇(5，7，4‘一trihydroxy一3‘一methyl一4一flavanol)。经M竹试验显示黄烯具有体外抑制He加细胞生长的作用，且具有更多还原

【Abstract】 Limonium bicolor Kunze (Bge.), commonly named as broomy grass and so on similarity the grass drought weeds in China, is a perennial (rare annual) herbaceous that belong to the genus of Limoniums (Plumbaginaceae) and grown in the sandiness or light salt region, such as grassland, meadow, mountainous region, is found in northeast, Yellow River drainage area province, and the north of Jiangsu, Mongolia and otherwise. The full plant is traditional drug, to heal bleeding, cancer nephritis and other weakness symptom. Several polyphenolic constituents Gallic acid, Myricetin-3-O-B-D-(6-galloyl)-galactoside, Quercetin, Eriodictyol, Luteolin, and have been isolated from aerial parts of Limonium bicolor, but till now no report related to the chemical composition and biological activities of it. It is well known that many biologically active polysaccharides have been isolated from vegetable kingdom; some of these polysaccharides are used clinically for cancer therapy. Flavones compounds, which generally exist as their glycosides in plants, are a kind of important secondary metabolites in plants. A lot of flavones showed interesting biological activities such as antioxidant, antibacterial, antivirus, and so on. Otherwise, the plant essential oils are other components, which generally have the biological activity of antibacterial, antioxidant, toxicity to pests. The present work is on the isolation, structural identification, and biological activity study of the polysaccharide, flavones and essential oil of Limonium bicolor. The main contents are summarized as follows:1 The recent progress on polysaccharides, flavones and essential oil, such as theisolation and purification, structural analysis, biological activities and structure-activity relationships of them were reviewed.2 Water-soluble polysaccharide (LP) was isolated from the aerial parts of the plants of Limonium bicolor by extraction with hot water. Purification by gel chromatogram of Sephadex. Analysis by gel penneation chromatography (GPC) showed it to be homogeneous and its molecular weight was about 4.63x103. From the results of acid hydrolysis, by GC-MS, FT-IR, 13C NMR, 1H NMR and element analysis, and so on, the result showed that glucose and glucose acid were the main components of LP, and the bond between GlcA and Glc was a-(1-6), then the disaccharide unit was connected by a-(l-4) linkage. The polysaccharides of LP had the fluorescence, 460nm was the highest emission peak, and the Zn2+ or Ca2+ enhanced the fluorescence intensity of LP. Much more research proved that LP had the activity of antioxidant and the function of inhibited the growth of Hela cells, with the IC50 was 62.2ug/mL.3 Two novel components had been isolated by solvent extraction and chromatography separation. The air-dried powder of the plant of Limonium bicolor were defatted with n-hexane and successively extracted with CHCl3, CHCl3-MeOH (9:1) and MeOH, in soxhlet apparatus, respectively. The methanolic residue was concentrated and chromatographed on Sephadex LH-20, then submitted to semi-preparative HPLC on a Eclipse XDB C18 column monitored by photo-diode-array detector, which led to the isolation of compound of 5,7,4’-trihydroxy-3’-methyl-flavan-4-ol, 5,7,4’-trihydroxy-4-methyl-2-flavene, along with other known polyphenolic compounds apigenin, methylgallate. The structure was identified by UV, FT-IR instrument, EI-MS, 1H NMR, 13C NMR spectrometer. The compound was the cytotoxic component in the extract with an IC50 of 3.9ug/ml against HeLa cells. The results of this investigation give support to the traditional use for treatment of tumor. The fluorescence spectra (Tris-HCl pH=7.4) showed its highest emission peak at 460nm, when exited at 388nm.4 The interaction of gallic acid-Zn (II) complex with DNA in solution was studied. The result showed that DNA could bind to calf thymus DNA in the presence of zinc ions, which have been confirmed by absorption, fluorescence spectra and HPLC. The increase of peak intensity and red更多还原